Antibody titres elicited by the 2018 seasonal inactivated influenza vaccine decline by 3 months post‐vaccination but persist for at least 6 months

Abstract Background In Australia, seasonal inactivated influenza vaccine is typically offered in April. However, the onset, peak and end of a typical influenza season vary, and optimal timing for vaccination remains unclear. Here, we investigated vaccine‐induced antibody response kinetics over 6 months in different age groups. Methods We conducted a prospective serosurvey among 71 adults aged 18–50 years, 15 community‐dwelling (‘healthy’) and 16 aged‐care facility resident (‘frail’) older adults aged ≥65 years who received the 2018 southern hemisphere vaccines. Sera were collected at baseline, and 1, 2, 4, and 6 months post‐vaccination. Antibody titres were measured by haemagglutination inhibition or microneutralisation assays. Geometric mean titres were estimated using random effects regression modelling and superimposed on 2014–2018 influenza season epidemic curves. Results Antibody titres peaked 1.2–1.3 months post‐vaccination for all viruses, declined by 3 months post‐vaccination but, notably, persisted above baseline after 6 months in all age groups by 1.3‐ to 1.5‐fold against A(H1N1)pdm09, 1.7‐ to 2‐fold against A(H3N2), 1.7‐ to 2.1‐fold against B/Yamagata and 1.8‐fold against B/Victoria. Antibody kinetics were similar among different age groups. Antibody responses were poor against cell‐culture grown compared to egg‐grown viruses. Conclusions These results suggest subtype‐specific antibody‐mediated protection persists for at least 6 months, which corresponds to the duration of a typical influenza season.

Conclusions: These results suggest subtype-specific antibody-mediated protection persists for at least 6 months, which corresponds to the duration of a typical influenza season. Annual influenza vaccination is recommended because the vaccine composition is updated each year to match circulating viruses and because antibody levels wane over time. 2 In Australia, IIV is usually available in March, timed to precede the start of the influenza season in April/May. Seasons typically peak in August and end in October, but there may be substantial variability. 3 For example, the 2017 influenza season started late but was associated with intense activity in primary care and hospitals, and widespread outbreaks in residential aged care facilities. 4 By contrast, the 2018 influenza season was characterised by low activity but was followed by unusually high levels of interseasonal activity in 2018/2019 and an early and prolonged 2019 season. 5 Such seasonal variation raises questions about optimal timing of influenza vaccination and whether vaccine-induced immunity is likely to persist for the duration of the season.
A post-vaccination geometric mean serum antibody titre ≥40, measured by haemagglutination inhibition (HI) assay, is the accepted correlate of protection for influenza vaccines, required by regulatory authorities for licencing. 6 However, age, 7 sex, 8 body mass index (BMI), 9 birth year 10 and influenza vaccination history 11 all influence the magnitude of the antibody response. Haemagglutinin (HA)-and neuraminidase (NA)-specific antibody levels peak around 2-6 weeks post-vaccination and decline to pre-season levels thereafter. [12][13][14] A systematic review and meta-analysis of antibody responses in older adults found consistent evidence for decay in titres from 21-42 days to 1 year post-vaccination. 2 Another review found titres did not decline faster in older adults compared with younger adults aged ≤65 years. 15 In most of the studies included in these analyses, titres were measured 1 and 6 months post-vaccination but not between. [16][17][18][19][20][21][22][23][24][25] The decline between 1 and 6 months has not been well characterised. Where more frequent intervals have been examined, they have been limited to community-dwelling 'healthy' older adults or 'frail' older adults without comparison of the two, 26 have not compared older and younger adults 27 or have not compared responses to quadrivalent (QIV) or enhanced trivalent vaccine in older adults. [28][29][30] In this prospective serosurvey, we collected sera from adults aged ≤50 years and older adults aged ≥65 years at baseline and 1, 2, 4 and 6 months post-vaccination to investigate the kinetics of antibody decay from 1 to 6 months. We superimposed our estimates on five influenza seasonal epidemic curves to investigate whether antibody levels persist for the duration of a typical influenza season. We also compared the kinetics between age groups, and between healthy and frail older adults. Additionally, vaccine antigens amplified in embryonated hens' eggs can acquire egg-adaptive mutations in the HA; therefore, we compared antibody titres against the egg-grown vaccine antigens with those against equivalent cell-culture grown viruses that lack egg-adaptive changes and are therefore more representative of clinical isolates. Participants were classified as (1) adults aged 18-50 years; (2) community-dwelling 'healthy' older adults aged ≥65 years or (3) 'frail' older adults aged ≥65 years. Eligibility was based on (i) being willing and able to provide informed consent; (ii) having no prior contraindications to the influenza vaccine; (iii) having no recent or current fever above 38 C; (iv) having no recent immunosuppressive treatment and (v) not having had already received the 2018 influenza vaccine. Age, sex, height, weight, medical history and vaccination history were collected at baseline; all data were self-reported.

| Vaccination
All participants received the 2018 southern hemisphere IIV appropriate for their age group and provided by their workplace or residence.

| Sample collection
Baseline blood samples were collected just prior to vaccination.
Follow-up samples were collected at 1, 2, 4 and 6 months post-vaccination. An additional sample was collected 12 months postvaccination from a subset of participants who could be contacted.

| Serological assays
Sera were treated with 600 μl receptor-destroying enzyme (RDE) (Denka-Seiken) per 200 μl serum, adsorbed with 5% turkey red blood cells (tRBC), and stored at 4 C. Sera were diluted twofold from 1:5 to 1:5120 to measure antibody titres to A(H1N1)pdm09, B/Yamagata and B/Victoria strains by HI assay using tRBC; titres against A(H3N2) strains were measured by microneutralisation (MN) assay. 31 Sera were tested against antigens that were included in the vaccines that the participants received. We compared antibody titres against the egg- Influenza B viruses were ether split. 32 HI titres were calculated as the reciprocal dilution of the last well with complete HI activity; MN titres were calculated as the reciprocal dilution of the last well where 50% of MDCK-SIAT cells were infected. The lower limit of detection for HI titres was 5 and for MN titres was 10; a negative result was assigned a value of half the lower limit of detection for calculation purposes.
Baseline, 1-, 2-, 4-and 6-month samples were measured in the same assay; 12-month samples were measured in an assay alongside baseline, 1-and 6-month samples. Seroconversion was defined as a ≥fourfold rise in titre and seropositivity by titres ≥40.

| Statistical analysis
All data cleaning, modelling and visualisation were completed in R version 3.5.1 (R Foundation for Statistical Computing, Vienna, Austria). Differences in baseline characteristics between groups were assessed using the Chi-square test for categorical outcomes and oneway ANOVA for continuous outcomes. The significance level was set at 0.05. Seropositivity was defined as a HI geometric mean titre (GMT) of at least 40; seroconversion was defined as a minimum fourfold rise in post-vaccination HI antibody titre. Average antibody decay was modelled as time since vaccination on log 2 HI titre using a linear mixed-effect model. 33 Using Akaike Information Criterion and prior assumptions about the antibody kinetic trajectory, the model included a natural cubic spline with three knots placed at quartiles. To compare decay curves between groups, an interaction term was included between time and group. Additional covariates (age and sex) were added to the model and their goodness of fit tested by likelihood ratio test. Predicted GMTs and 95% prediction intervals (95%PI) were estimated using a semi-parametric bootstrap for mixed models. 33 Differences in antibody decay curves were estimated across groups by type III ANOVA (using Satterthwaite's degrees of freedom method 34 ).
Older adult participants received e-TIV that did not contain a B/Victoria component, so their antibody titres measured against B/Victoria were not included in analyses. Log 2 HI titres were backtransformed for ease of interpretation.
Epidemic curves were produced using National Notifiable Diseases Surveillance System (NNDSS) notification data from the state of Victoria. These were smoothed using a 3-week moving average and superimposed on predicted antibody titre curves.

| Post-vaccination antibody titres against influenza vaccine antigens and the equivalent cell-culture grown viruses by age group
Antibody titres were measured against each antigen contained in the influenza vaccine and against the equivalent cell-culture grown viruses ( Figure 2).
In all age groups, predicted GMTs were higher against the egg-grown viruses than against the equivalent cell-culture grown

| Antibody response by sex
Among the adult group, antibody titres did not significantly differ by sex, except against the B/Yamagata vaccine antigen where titres were higher in males than females (p = 0.02), though the rate of antibody decay against this antigen did not differ (p = 0.72) (data not shown).

| DISCUSSION
Overall, this study shows that the influenza vaccine-induced antibody response declined after 3 months but importantly persisted above gen; however, we generally did not observe any difference between age groups or by sex.
Notably, our study fills a gap in the literature by defining the timeframe wherein antibody titres begin to decline; earlier studies could only infer that the decline occurs between 1 and 6 months postvaccination. [16][17][18][19][20][21][22][23][24][25] We also compared titres between healthy young adults and healthy and frail older adults using licenced QIV and eTIV in Australia. [28][29][30] Our finding that antibody titres persisted above baseline for at least 6 months, and even up to 12 months post-vaccination against influenza B antigens, is consistent with a previous study that showed anti-influenza HA and NA antibody titres in younger adults remained high after 18 months and may even persist over multiple seasons. 35 A study in older adults observed sustained titres over a 12-month period following IIV, 25  We observed that the kinetics of antibody decay did not differ between adults aged 18-50 and healthy and frail older adults aged ≥65. This is consistent with a meta-analysis that suggested antibody levels following influenza vaccination in older adults do not necessarily decline at a higher rate than younger adults as is commonly believed. 15  however, there was little difference in the rate of antibody decline between 1 and 6 months. 24 While we cannot make a clear comparison in our cohort, there may be minimal difference between the kinetics of the antibody response following standard-dose and high-dose IIV in older adults.
We observed that rates of seroconversion against influenza vaccine antigens were consistently greatest in the frail older adult group. Nunez et al. 37 also observed higher seroconversion rates to influenza A in an older age group compared to a younger age group, and they noted that this was dependent on seropositivity F I G U R E 3 Differences in antibody response to 4 H3N2 reference antigens. Sera were collected from participants in different age groups at baseline and 1, 2, 4 and 6 months post-vaccination. Serum antibody titres were measured by microneutralisation assay against cell-culture grown A(H3N2) strains in four different A(H3N2) clades. Lines show the predicted geometric mean titres (GMT); shaded areas show 95% prediction intervals (95%PI); points represent individual titre values. Points are jittered.
status where HI titres <40 are considered 'seronegative' prior to vaccination. Individuals were not more or less likely to seroconvert based on age, but younger individuals were more likely to be seropositive prior to vaccination and therefore remain seropositive, without seroconverting. 37 Antibody responses to the cell-culture grown equivalent strains for all subtypes were equal to or lower than against the vaccine antigens in all age groups tested. The greatest difference in titres was observed between cell-culture and egg-grown A(H3N2) strains, and far fewer people seroconverted against the cell-grown A(H3N2) virus than against the vaccine antigen. Furthermore, we observed consistently poor responses to cell-grown A(H3N2) strains from antigenically distinct clades. These findings are consistent with a report of altered antibody titres between A(H3N2) vaccine strains and circulating viruses, 38 which also noted that titres against circulating but not  40,42 This is one of the causes of sub-optimal vaccine effectiveness against A(H3N2) in recent years. [43][44][45] Poor vaccine effectiveness against A(H3N2) has also been attributed to mismatch between vaccine and circulating strains. 46,47 Influenza viruses continuously evolve leading to substantial diversification of circulating A(H3N2) viruses 48 with several antigenically distinct groups co-circulating, making it difficult to select vaccine candidates that afford broad coverage.
While previous studies have suggested that the antibody response tends to be higher in females than in males, 49 50 We did not monitor participants for influenza infection during the study which may interfere with antibody kinetics. We also were unable to examine the effects of obesity that has been associated with higher initial rises, but steeper declines in titre. 9 Finally, we were unable to compare responses to high dose or adjuvanted eTIVs in the healthy and frail older adults due to insufficient numbers and the observational nature of this study.
While the optimal timing for influenza vaccination remains uncertain, we found that antibody responses following influenza vaccination wane over time but, crucially, persist for the duration of a typical influenza season. Further research is required before changes to current policy regarding the timing for influenza vaccine rollout should be recommended. The consequences of delaying vaccination on overall vaccine uptake, immunological factors, logistical challenges of condensed vaccine rollout while achieving the same level of coverage, changes to the manufacturing process and inter-season variability must all first be carefully considered. Our findings, taken together with the wide range of uncertainties regarding the optimal timing for influenza vaccination, suggest any policy changes at this stage may be premature.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1111/irv.13072.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.